The purpose of this proposal is to continue our study of the role of the enzyme superoxide dismutase (SOB) in cancer. This protective enzyme has been found, in general, to be low in tumor cells when compared to an appropriate control. We propose in this application to study at the molecular level why this enzyme is diminished in tumor cells and to determine what, if any, significance this loss has to the cancer cell phenotype. Work will be continued on two in vitro cell systems. In these systems four enzymes will be studied: copper and zinc-containing SOD (CuZnSOD), manganese-containing SOD (MnSOD), catalase (CAT), and glutathione peroxidase (GPX). The following will be measured: enzyme activity, immunoreactive protein using Western blotting, mRNA for the antioxidant enzymes using Northern blotting and in vitro translation, and gene copy number and gross gene rearrangements using Southern blotting. To determine the significance of the loss of SOD in tumor cells, transfection experiments with MnSOD cDNA will be performed. An expression vector for sense MnSOD cDNA will be transfected into tumor cells and clones that stably express high MnSOD isolated. These clones will then be tested for malignant properties. Immortal, non-malignant cells will also be transfected with sense MnSOD cDNA and clones with high MnSOD isolated. Clones expressing high MnSOD will then be tested for susceptibility to transformation and the results compared to controls. Similar experiments will be performed with non-differentiating, non-malignant cells to determine if expression of MnSOD allows differentiation to proceed. Likewise, a vector that contains the antisense cDNA for MnSOD will be transfected into normal cells and the effect on the phenotype studied. Cells transfected with antisense MnSOD cDNA will also be studied for susceptibility to carcinogenesis and for differentiation potential. These experiments should define the role of MnSOD malignancy. If MnSOD is found to be responsible for part of the cancer cell phenotype, modulation of this enzyme might be useful in preventing or treating cancer.